Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy

A dysfunction of amino acid neurotransmitter transporters occurs in a number of central nervous system disorders, including stroke, epilepsy, cerebral palsy and amyotrophic lateral sclerosis. This dysfunction can comprise a reversal of transport direction, leading to the release of neurotransmitter into the extracellular space, or an alteration in transporter expression level. This review analyses the role of glutamate and GABA transporters in the pathogenesis and therapy of a number of acute and chronic neurological disorders.     

Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors

Neurotransmitter transporters can affect neuronal excitability indirectly via modulation of neurotransmitter concentrations or directly via transporter currents. A physiological or pathophysiological role for transporter currents has not been described. We found that GABA transporter 1 (GAT-1) cation currents directly increased GABAergic neuronal excitability and synaptic GABA release in the periaqueductal gray (PAG) during opioid withdrawal in rodents. In contrast, GAT-1 did not indirectly alter GABA receptor responses via modulation of extracellular GABA concentrations. Notably, we found that GAT-1-induced increases in GABAergic activity contributed to many PAG-mediated signs of opioid withdrawal. Together, these data support the hypothesis that GAT-1 activity directly produces opioid withdrawal signs through direct hyperexcitation of GABAergic PAG neurons and nerve terminals, which presumably enhances GABAergic inhibition of PAG output neurons. These data provide, to the best of our knowledge, the first evidence that dysregulation of a neurotransmitter transporter current is important for the maladaptive plasticity that underlies opiate withdrawal.     

Vigabatrin induces tonic inhibition via GABA transporter reversal without increasing vesicular GABA release

Two forms of GABAergic inhibition coexist: fast synaptic neurotransmission and tonic activation of GABA receptors due to ambient GABA. The mechanisms regulating ambient GABA have not been well defined. Here we examined the role of the GABA transporter in the increase in ambient [GABA] induced by the anticonvulsant vigabatrin. Pretreatment of cultured rat hippocampal neurons with vigabatrin (100 microM) for 2-5 days led to a large increase in ambient [GABA] that was measured as the change in holding current induced by bicuculline during patch-clamp recordings. In contrast, there was a decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents mIPSCs with no change in their amplitude distribution, and a decrease in the magnitude of IPSCs evoked by presynaptic stimulation during paired recordings. The increase in ambient [GABA] was not prevented by blockade of vesicular GABA release with tetanus toxin or removal of extracellular calcium. During perforated patch recordings, the increase in ambient [GABA] was prevented by blocking the GABA transporter, indicating that the GABA transporter was continuously operating in reverse and releasing GABA. In contrast, blocking the GABA transporter increased ambient [GABA] during whole cell patch-clamp recordings unless GABA and Na(+) were added to the recording electrode solution, indicating that whole cell recordings can lead to erroneous conclusions about the role of the GABA transporter in control of ambient GABA. We conclude that the equilibrium for the GABA transporter is a major determinant of ambient [GABA] and tonic GABAergic inhibition. We propose that fast GABAergic neurotransmission and tonic inhibition can be independently modified and play complementary roles in control of neuronal excitability.     

Transport rates of GABA transporters: regulation by the N-terminal domain and syntaxin 1A

Plasma membrane GABA transporters participate in neural signaling through re-uptake of neurotransmitter. The domains of the transporter that mediate GABA translocation and regulate transport are not well understood. In the present experiments, the N-terminal cytoplasmic domain of the GABA transporter GAT1 regulated substrate transport rates. This domain directly interacted with syntaxin 1A, a SNARE protein involved in both neurotransmitter release and modulation of calcium channels and cystic fibrosis transmembrane regulator (CFTR) chloride channels. The interaction resulted in a decrease in transporter transport rates. These data demonstrate that intracellular domains of the GABA and protein-protein interactions regulate substrate translocation, and identify a direct link between the machinery involved in transmitter release and re-uptake.     

Synaptically evoked GABA transporter currents in neocortical glia

The presence, magnitude, and time course of GABA transporter currents were investigated in electrophysiologically characterized neocortical astrocytes in an in vitro slice preparation. On stimulation with a bipolar-tungsten stimulating electrode placed nearby, the majority of cells tested displayed long-lasting GABA transporter currents using both single and repetitive stimulation protocols. Using subtype-specific GABA transporter antagonists, long-lasting GABA transporter currents were identified in neocortical astrocytes that originated from at least two subtypes of GABA transporters: GAT-1 and GAT-2/3. These transporter currents displayed slow rise times and long decay times, contrasting the time course observed for glutamate transporter currents, and are indicative of a long extracellular time course of GABA as well as a role for glial GABA transporters during synaptic transmission.     

The SLC32 transporter,a key protein for the synaptic release of inhibitory amino acids

The SLC32 family comprises a single member: the vesicular inhibitory amino acid transporter (VIAAT) or vesicular GABA transporter (VGAT). It belongs to a eukaryotic-specific superfamily of H(+)-coupled amino acid transporters, which also comprises the mammalian SLC36 and SLC38 transporters. VIAAT exchanges GABA or glycine for protons. It is present on synaptic vesicles of GABAergic and glycinergic neurons, and in some endocrine cells, where it ensures the H(+)-ATPase-driven uptake, and subsequent exocytotic release, of inhibitory amino acids. Despite a similar function in vesicular neurotransmitter loading, VIAAT is not related to the vesicular glutamate transporter (VGLUT, SLC17) or the vesicular monoamine transporter/vesicular acetylcholine transporter (VMAT/VACHT, SLC18) proteins.     

Characterization of gamma-aminobutyric acid responses with sulfate loading in cat bladder neurons

In some cells in cat bladder ganglia gamma-aminobutyric acid (GABA) applied iontophoretically produced a hyperpolarizing response accompanied by an increase in input conductance when recorded with potassium sulfate-filled microelectrodes. This GABA-induced hyperpolarization was blocked by bicuculline and was converted to a depolarizing GABA response when extracellular chloride concentration was low suggesting that the hyperpolarizing GABA response was mediated by the opening of chloride channels. In other cells, continuous passage of a small negative current converted a depolarizing GABA response to a hyperpolarizing response with time. This effect was accompanied by a negative shift of the reversal potential. These data indicated that injection of impermeable sulfate ions decreased the intracellular chloride concentration.     

GABA uptake and heterotransport are impaired in the dentate gyrus of epileptic rats and humans with temporal lobe sclerosis

In vivo dialysis and in vitro electrophysiological studies suggest that GABA uptake is altered in the dentate gyrus of human temporal lobe epileptics characterized with mesial temporal sclerosis (MTLE). Concordantly, anatomical studies have shown that the pattern of GABA-transporter immunoreactivity is also altered in this region. This decrease in GABA uptake, presumably due to a change in the GABA transporter system, may help preserve inhibitory tone interictally. However, transporter reversal can also occur under several conditions, including elevations in [K(+)]o, which occurs during seizures. Thus GABA transporters could contribute to seizure termination and propagation through heterotransport. To test whether GABA transport is compromised in both the forward (uptake) and reverse (heterotransport) direction in the sclerotic epileptic dentate gyrus, the physiological effects of microapplied GABA and nipecotic acid (NPA; a compound that induces heterotransport) were examined in granule cells in hippocampal slices from kainate (KA)-induced epileptic rats and patients with temporal lobe epilepsy (TLE). GABA- and NPA-induced responses were prolonged in granule cells from epileptic rats versus controls (51.3 and 31.3% increase, respectively) while the conductance change evoked with NPA microapplication was reduced by 40%. Furthermore the ratio of GABA/NPA conductance, but not duration, was significantly >1 in epileptic rats but not controls, suggesting a compromise in transporter function in both directions. Similar changes were observed in tissue resected from epileptic patients with medial temporal sclerosis but not in those without the anatomical changes associated with MTLE. These data suggest that the GABA transporter system is functionally compromised in both the forward and reverse directions in the dentate gyrus of chronically epileptic tissue characterized by mesial temporal sclerosis. This alteration may enhance inhibitory tone interically yet be permissive for seizure propagation due to a decreased probability for GABA heterotransport during seizures.     

Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.     

Dopamine transporter-mediated conductances increase excitability of midbrain dopamine neurons

Uptake by Na(+)/Cl(-)-dependent neurotransmitter transporters is the principal mechanism by which extracellular biogenic amine concentrations are regulated. In addition to uptake, the cloned transporter proteins also elicit ion channel-like currents, but the physiological consequences of these currents are unknown. Here, whole-cell patch clamp and perforated-patch recordings show that substrates of the dopamine transporter (DAT), such as dopamine (DA) and amphetamine, increase the firing activity of rat DA neurons in culture. We found that these substrates elicit inward currents that are Na(+)-dependent and blocked by cocaine. These currents are primarily comprised of anions and result in an excitatory response in DA neurons at lower DA concentrations than are required for D2 autoreceptor activation. Thus, in addition to clearing extracellular DA, our results suggest that the currents associated with DAT modulate excitability and may regulate release of neurotransmitter from midbrain DA neurons.     

Upregulation of the GABA transporter GAT-1 in the gracile nucleus in the spared nerve injury model of neuropathic pain

Neuropathic pain is a major health issue and is frequently accompanied by allodynia (painful sensations in response to normally non-painful stimulations), and unpleasant paresthesia/dysesthesia, pointing to alterations in sensory pathways normally dedicated to the processing of non-nociceptive information. Interestingly, mounting evidence indicate that central glial cells are key players in allodynia, partly due to changes in the astrocytic capacity to scavenge extracellular glutamate and γ-aminobutyric acid (GABA), through changes in their respective transporters (EAAT and GAT). In the present study, we investigated the glial changes occurring in the dorsal column nuclei, the major target of normally innocuous sensory information, in the rat spared nerve injury (SNI) model of neuropathic pain. We report that together with a robust microglial and astrocytic reaction in the ipsilateral gracile nucleus, the GABA transporter GAT-1 is upregulated with no change in GAT-3 or glutamate transporters. Furthermore, [³H] GABA reuptake on crude synaptosome preparation shows that transporter activity is functionally increased ipsilaterally in SNI rats. This GAT-1 upregulation appears evenly distributed in the gracile nucleus and colocalizes with astrocytic activation. Neither glial activation nor GAT-1 modulation was detected in the cuneate nucleus. Together, the present results point to GABA transport in the gracile nucleus as a putative therapeutic target against abnormal sensory perceptions related to neuropathic pain.     

Molecular regulation of glutamate and GABA transporter proteins by valproic acid in rat hippocampus during epileptogenesis

Epileptiform discharges and behavioral seizures may be the consequences of the presence of either excessive excitation associated with the neurotransmitter glutamate or from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove these amino acids from the synaptic cleft. The glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino acids carrier-1 (EAAC-1) limit excitation initiated by synaptic release of glutamate. Transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3) remove GABA from synaptic regions. To assess the molecular effects of the antiepileptic drug valproate, albino rats with chronic, spontaneous, recurrent seizures induced by amygdalar injection of FeCl3 were treated for 14 days with either valproic acid or with saline as an injection control. Regions of the hippocampus were assayed for glutamate and GABA transporters by western blot. While epileptogenesis is thought to correlate with the downregulation of GLAST and upregulation of EAAC-1, valproate caused an increase in the quantity of GLAST protein measured in the hippocampus. Valproate treatment decreased GLT-1 in both control and experimental animals in both hippocampi. EAAC-1 was unchanged by valproate treatment. GABA transporters GAT-1 and GAT-3 in the hippocampus were upregulated by FeCl3 injection into the amygdala. However, valproate caused the downregulation of these GABA transporters in both control and experimental animals. Altered molecular regulation of glutamate appears to be critical in the development of sustained, spontaneous limbic seizures. Our data suggest that valproate may have unique mechanisms of action; specifically, it may affect the removal of glutamate by upregulating GLAST and decreasing GABA transport, which could result in increased tissue concentrations of GABA.     

Amygdala kindling induces upregulation of mRNA for NKCC1, a Na(+), K(+)-2Cl(-) cotransporter,in the rat piriform cortex

GABA, the main inhibitory neurotransmitter in the brain, elicits a hyperpolarizing response by activation of the GABA(A)-receptor/chloride-channel complex under conditions of normal Cl(-) homeostasis. Thus the pathogenesis of epilepsy could involve an impairment of GABA(A)-receptor-mediated inhibition due to a collapse of the Cl(-) gradient. We examined the expression patterns of Cl(-) transporters and a Cl(-) channel in a rat amygdala-kindling model. Activity-dependent increases were observed in the mRNA for NKCC1, an inwardly-directed Cl(-) transporter, in the piriform cortex. This suggests that an increase in [Cl(-)](i) and a resultant reduction in GABAergic inhibition may occur in the kindled piriform cortex.     

GABA transporter-1 (GAT1)-deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus

After its release from interneurons in the CNS, the major inhibitory neurotransmitter GABA is taken up by GABA transporters (GATs). The predominant neuronal GABA transporter GAT1 is localized in GABAergic axons and nerve terminals, where it is thought to influence GABAergic synaptic transmission, but the details of this regulation are unclear. To address this issue, we have generated a strain of GAT1-deficient mice. We observed a large increase in a tonic postsynaptic hippocampal GABAA receptor-mediated conductance. There was little or no change in the waveform or amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) or miniature IPSCs. In contrast, the frequency of quantal GABA release was one-third of wild type (WT), although the densities of GABAA receptors, GABAB receptors, glutamic acid decarboxylase 65 kDa, and vesicular GAT were unaltered. The GAT1-deficient mice lacked a presynaptic GABAB receptor tone, present in WT mice, which reduces the frequency of spontaneous IPSCs. We conclude that GAT1 deficiency leads to enhanced extracellular GABA levels resulting in an overactivation of GABAA receptors responsible for a postsynaptic tonic conductance. Chronically elevated GABA levels also downregulate phasic GABA release and reduce presynaptic signaling via GABAB receptors thus causing an enhanced tonic and a diminished phasic inhibition.     

Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization

Neurotransmitter transporters are critical for synaptic neurotransmitter inactivation. Transporter inhibitors markedly increase the duration and magnitude of synaptic transmission, underscoring the importance of transporter activity in neurotransmission. Recent studies indicate that membrane trafficking dynamically governs neuronal transporter cell-surface presentation in a protein kinase C-regulated manner, suggesting that transporter trafficking profoundly affects synaptic signaling. However, the molecular architecture coupling neurotransmitter transporters to the endocytic machinery is not defined. Here, we identify nonclassical, distinct endocytic signals in the dopamine transporter (DAT) that are necessary and sufficient to drive constitutive and protein kinase C-regulated DAT internalization. The DAT internalization signal is conserved across SLC6 neurotransmitter carriers and is functional in the homologous norepinephrine transporter, suggesting that this region is likely to be the endocytic signal for all SLC6 neurotransmitter transporters. The DAT endocytic signal does not conform to classic internalization motifs, suggesting that SLC6 neurotransmitter transporters may have evolved unique endocytic mechanisms.     

Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl- in hippocampal CA3 neurons

1. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells in organotypic hippocampal slice cultures for measurement of membrane currents underlying both the gamma-aminobutyric acid (GABA)-mediated, Cl- -dependent inhibitory postsynaptic potential (IPSC), evoked in response to stimulation of the mossy fiber pathway, and responses to iontophoretically applied GABA. Their reversal potentials are presumed to equal the equilibrium potential for Cl- (37). Mechanisms underlying activity-dependent increases in the intracellular concentration of Cl- ([Cl-]i) were investigated by describing active and passive pathways for Cl- influx and efflux. 2. During 99-s applications of GABA, driving force declined by 51% due to increases in [Cl-]i; thus passive Cl- influx through GABA-activated pathways can significantly affect [Cl-]i. 3. Decreasing the extracellular K+ concentration ([K+]o) from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean IPSC reversal potential (EIPSC) from -67.6 to -81.9 mV, even when membrane potential (Vm) was maintained constant and depolarized with respect to EIPSC. 4. Decreasing [K+]o from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean GABA reversal potential (EGABA) from -64.7 to -81.1 mV, even when Vm was maintained constant and depolarized with respect to EGABA. Reducing the extracellular Cl- concentration from 153 to 89 mM, while maintaining [K+]o constant at 1 mM, shifted the mean EGABA from -81.1 to -66.2 mV, an amount close to that predicted by the Nernst equation for Cl-. We conclude that reducing [K+]o caused a hyperpolarizing shift in EGABA and EIPSC by decreasing [Cl-]i. 5. The shift of EIPSC and EGABA upon alteration of [K+]o did not result from contamination of the responses by additional K+-mediated components because it was unaffected by block of K+ channels with intracellular Cs+. 6. Reducing the extracellular Na+ concentration from 141 to 70 mM had no effect on EGABA. 7. Furosemide, bath-applied at 5 X 10(-4) M while holding Vm depolarized with respect to EIPSC, caused a rapid, reversible decrease in IPSC driving force averaging 69%, consistent with the presence of a furosemide-sensitive outward Cl- -transport system. 8. Reducing [K+]o from 5.8 to 1 mM in the presence of 5 X 10(-4) M furosemide produced a smaller shift of EIPSC from -61.0 to -71.2 mV, however, after washout of furosemide from [K+]o = 1 mM saline, EIPSC shifted further to -89.8 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular chloride concentration is higher in rod bipolar cells than in cone bipolar cells of the mouse retina

Bipolar cells (BCs) have antagonistic center-surround receptive field. Surround illumination evokes depolarization in the OFF-type cone BC, and hyperpolarization in the rod BC and the ON-type cone BC. Surround illumination reduces gamma-aminobutyric acid (GABA) release from horizontal cells. If GABA hyperpolarize BCs, the polarity of the GABA-induced effect agrees with the light-evoked surround response in the OFF-type BC, but contradicts in the rod BC and the ON-type cone BC. Immunohistochemical study of the Cl⁻ transporter of BCs has suggested that the intracellular Cl⁻ concentration is different among BC subtypes. We examined the reversal potential of GABA-induced current of BCs using gramicidin-perforated patch clamp technique in the mouse retina, and found that GABA depolarizes rod BC and hyperpolarizes cone BCs. Our results are consistent with the GABAergic input to rod BC dendrite.     

Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6

The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins.